Arenaviruses cause acute hemorrhagic fevers with high mortality worldwide. Junn virus is recognized as a category A pathogen and Material Threat to the US population. Effective therapies against arenaviral hemorrhagic fevers are urgently needed to address ongoing public health and biodefense concerns. Our long- term goal is to elucidate the molecular mechanisms by which Junn virus appropriates specific cellular functions to implement its RNA-based replicative cycle in the host-cell cytoplasm. We have recently identified unique cytoplasmic structures that serve as compartments for arenavirus replication and transcription (RTCs) and may also be involved in viral translation. Our overarching hypothesis is that novel activities of the virus nucleoprotein are essential for induction of these membrane-associated RTCs and for preferential recruitment of ribosomes to viral mRNAs. In this proposal, we will define determinants in the multifunctional nucleoprotein that interface with the cellular infrastructure to organize an environment permissive for viral replication. This knowledge will increase our understanding of arenavirus biology and suggest novel antiviral strategies for the treatment of acute hemorrhagic fevers. Our Specific Aims are to: (1) Investigate the origins, structure and function of RTCs by systematically identifying co-localizing cellular components. Our preliminary results suggest that RTCs comprise a unique combination of cellular elements from both membrane-remodeling and translational-control pathways. Microscopic and biochemical analysis of the co-opted proteins and pathways will establish a framework for understanding fundamental virus- cell interactions required for arenavirus replication. (2) Characterize the functional role of nucleoprotein in ribosome recruitment and viral translation. Confocal microscopy studies reveal that RTCs contain various translation initiation factors and ribosomes, but are specifically lacking the cap-binding protein eIF4E. We posit that N acts as a surrogate to bind m7G-capped viral mRNA and recruit the eIF4G scaffold protein, in order to gain privileged access to ribosomes. We will employ biochemical and pharmacological approaches to determine the functional requirement for nucleoprotein in viral translation. Knowledge gained from these studies will be important in guiding the development of small-molecule compounds to inhibit this novel activity. We anticipate that essential virus-cell interactions can be targeted to generate highly specific antiviral effects that are resistant to genetic escape. PUBLIC HEALTH RELEVANCE: Arenaviruses cause acute hemorrhagic fevers with high mortality worldwide. Effective therapies are urgently needed to address ongoing public health and biodefense concerns. Viruses depend extensively on interactions with the host cell, and this project will characterize novel functions of the viral nucleoprotein required for viral replication transcription and translation. The results from these studies will increase our understanding of arenavirus biology and suggest novel antiviral strategies for the treatment of arenavirus hemorrhagic fevers.